Lipoyl compounds and their use for treating ischemic injury

ABSTRACT

The present invention relates, in various embodiments, to a compound represented by Structural Formula (I), pharmaceutically acceptable salts or prodrugs thereof, and compositions comprising said compounds, or pharmaceutically acceptable salts or prodrugs thereof. Methods of using compounds of Structural Formulas (I) and (la) or compositions comprising compounds of Structural Formulas (I) and (la), or pharmaceutically acceptable salts or prodrugs thereof, to treat ischemia or ischemia-reperfusion injury are also disclosed.

RELATED APPLICATIONS

This application is the U.S. National Stage of International ApplicationNo. PCT/US2011/060259, filed Nov. 10, 2011, which designates the U.S.,published in English, and claims the benefit of U.S. ProvisionalApplication No. 61/415,240, filed on Nov. 18, 2010; U.S. ProvisionalApplication No. 61/415,241, filed on Nov. 18, 2010; U.S. ProvisionalApplication No. 61/478,310, filed on Apr. 22, 2011; and U.S. ProvisionalApplication No. 61/500,974, filed on Jun. 24, 2011. The entire contentsof the above applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Coronary heart disease is a leading cause of death and injury worldwide.Following an acute myocardial infarction (MI), early restoration ofblood flow is the most effective strategy for reducing the size of theMI. Paradoxically, restoring blood flow to the area of the heart that isaffected by the diminished flow can, in itself, be harmful. This iscalled reperfusion injury and can, by some estimates, be responsible forup to 50% of the final size of the MI (Yellon D. M., Hausenloy D. J.,Myocardial reperfusion injury. New England Journal of Medicine 2007,357:1121). The final size of the MI ultimately determines how well theheart can function after a heart attack.

Myocardial ischemia-reperfusion injury is defined as myocardial injurycaused by the ischemic injury combined with injury caused by therestoration of coronary blood flow after an ischemic episode.Ischemia-reperfusion injury is mediated by an influx of calcium ions anddepletion of oxygen during an ischemic event, followed by reoxygenationand generation of reactive oxygen species during reperfusion (Piper, H.M., Abdallah, C., Schafer, C., The first minutes of reperfusion: awindow of opportunity for cardioprotection. Annals of Thoracic Surgery2003, 75:644; Yellon, D. M., Hausenloy, D. J., Myocardial reperfusioninjury. New England Journal of Medicine 2007, 357:1121). It ispostulated that the influx of calcium and the increase in free radicalstriggers cell death, or programmed cell death (Chen, X., Zhang, X.,Hubo, H., et al., Ca²⁺ influx-induced sarcoplasmic reticulum Ca²⁺overload causes mitochondrial-dependent cell death in ventricularmyocytes. Circ Res 2005, 97:1009; Lopes-Neblina, F., Toledo, A. H.,Toledu-Pereyra, L. H. Molecular biology of apoptosis in ischemia andreperfusion. J Invest Surg 2005, 18:335). However, treatment of patientswith acute myocardial infarction with antagonists that block the influxof calcium or with scavengers of the reactive oxygen species has yieldeddisappointing clinical outcomes (Yellon, D. M., Hausenloy, D. J.,Myocardial reperfusion injury. New England Journal of Medicine 2007,357:1121).

Another strategy for reducing ischemia-reperfusion injury is termedischemic preconditioning. Short repeated bouts of ischemia followed byreperfusion will condition the myocardium to withstand a prolonged boutof ischemia (Otani, H., Ischemic preconditioning: From moleculemechanisms to therapeutic opportunities. Antioxidants & Redox Signaling,2008, 10:207). However, intentionally occluding a patient's coronaryartery is associated with undue risks and is therefore undesirable.

Thus, there is a significant need for new and more effective therapiesand therapeutic agents for the treatment of ischemia andischemia-reperfusion injuries resulting from cardiovascular disease andother conditions.

SUMMARY OF THE INVENTION

The invention described herein addresses a need for treating ischemia,ischemic injury and ischemia-reperfusion injury, including myocardialischemia. In particular, the present invention relates to compositionscomprising the disclosed compounds, or pharmaceutically acceptable saltsor prodrugs thereof, and methods of using the disclosed compounds, orpharmaceutically acceptable salts or prodrugs thereof, to treatischemia, ischemic injury, ischemia-reperfusion injury, and relatedconditions.

The compounds of the present invention are represented by StructuralFormula (I):

or a pharmaceutically acceptable salt or prodrug thereof, substantiallyseparated from the (S)-lipoyl stereoisomer(s), or a pharmaceuticallyacceptable salt or prodrug thereof wherein:

-   R is (C₁-C₁₈)alkyl, (C₆-C₁₈)aryl or (C₆-C₁₈)aryl(C₁-C₁₈)alkyl and is    substituted with at least one acidic substituent selected from the    group consisting of —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H, —OPO₃H₂, —B(OH)₂    and —NHOH, wherein the aryl of the (C₆-C₁₈)aryl or    (C₆-C₁₈)aryl(C₁-C₁₈)alkyl is optionally further substituted with one    or more substituents selected from the group consisting of hydroxy,    halo, (C₁-C₃)alkyl, halo(C₁-C₃)alkyl, cyano, nitro, (C₁-C₃)alkoxy    and thio(C₁C₃)alkyl;-   R′ is hydrogen or (C₁-C₁₈)alkyl, wherein (C₁-C₁₈)alkyl is optionally    substituted with one or more acidic substituents selected from the    group consisting of —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H, −OPO₃H₂, —B(OH)₂    and —NHOH; and-   X is absent or is an amino acid, wherein the amino acid is oriented    to form an amide linkage with

provided that the compound of Structural Formula (I) is notN—(R)-lipoyl-glutamylalanine, N—(R)-lipoyl-aminoethylphosphonic acid, or(R)-5-(5-(1,2-dithiolan-3-yl)pentanamido)-2-hydroxybenzoic acid.

The present invention also provides a method of treating ischemic injuryor ischemia-reperfusion injury in a subject in need thereof, comprisingadministering to the subject an effective amount of a compoundrepresented by Structural Formula (Ia):

or a pharmaceutically acceptable salt or prodrug thereof, wherein:

-   R is (C₁-C₁₈)alkyl, (C₆-C₁₈)aryl or (C₆-C₁₈)aryl(C₁-C₁₈)alkyl and is    substituted with at least one acidic substituent selected from the    group consisting of —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H, —OPO₃H₂, —B(OH)₂    and —NHOH, wherein the aryl of the (C₆-C₁₈)aryl or    (C₆-C₁₈)aryl(C₁-C₁₈)alkyl is optionally further substituted with one    or more substituents selected from the group consisting of hydroxy,    halo, (C₁-C₃)alkyl, halo(C₁-C₃)alkyl, cyano, nitro, (C₁-C₃)alkoxy    and thio(C₁-C₃)alkyl;-   R′ is hydrogen or (C₁-C₁₈)alkyl, wherein (C₁-C₁₈)alkyl is optionally    substituted with one or more acidic substituents selected from the    group consisting of —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H, —OPO₃H₂, —B(OH)₂    and —NHOH; and-   X is absent or is an amino acid, wherein the amino acid is oriented    to form an amide linkage with

provided that the compound of Structural Formula (Ia) is notN-lipoyl-glutamylalanine, N-lipoyl-aspartylglycine,N-lipoyl-glutamylglycine or5-(5-(1,2-dithiolan-3-yl)pentanamido)-2-hydroxybenzoic acid.

In another embodiment, the invention relates to a method of inhibitingcell death in a subject, comprising administering to the subject aneffective amount of a compound represented by Structural Formula (I)and/or (Ia), or a pharmaceutically acceptable salt or prodrug thereof.

In yet another embodiment, the invention relates to a method ofinhibiting cell death in a cell, tissue or organ, wherein the cell,tissue or organ has experienced an ischemia or other condition ordisorder that results in excessive or unwanted cell death, comprisingadministering to the cell, tissue or organ an effective amount of acompound represented by Structural Formula (I) and/or (Ia), or apharmaceutically acceptable salt or prodrug thereof.

Also included in the present invention is the use of a compoundrepresented by Structural Formula (I) and/or (Ia), or a pharmaceuticallyacceptable salt or prodrug thereof, for treating ischemic injury orischemia-reperfusion injury in a subject.

In another embodiment, the invention relates to the use of a compoundrepresented by Structural Formula (I) and/or (Ia), or a pharmaceuticallyacceptable salt or prodrug thereof, for inhibiting cell death in asubject.

The present invention also includes the use of a compound represented byStructural Formula (I) and/or (Ia), or a pharmaceutically acceptablesalt or prodrug thereof, for the manufacture of a medicament fortreating ischemic injury or ischemia-reperfusion injury in a subject.

The invention also includes the use of a compound represented byStructural Formula (I) and/or (Ia), or a pharmaceutically acceptablesalt or prodrug thereof, for the manufacture of a medicament forinhibiting cell death in a subject.

The compounds (also referred to herein as “the disclosed compounds”),compositions and methods of the present invention are efficacious fortreating tissue damage resulting from ischemia and ischemic injuries,including ischemia-reperfusion injuries.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The disclosed compounds may exist in various stereoisomeric forms unlessotherwise specified. “Stereoisomers” are compounds that differ only intheir spatial arrangement. “Enantiomers” are pairs of stereoisomers thatare non-superimposable mirror images of one another, most commonlybecause they contain an asymmetrically substituted carbon atom that actsas a chiral center.

“Diastereomers” are stereoisomers that are not related as mirror images,most commonly because they contain two or more asymmetricallysubstituted carbon atoms. “R” and “S” represent the configuration ofsubstituents around one or more chiral carbon atoms.

“Racemate” or “racemic mixture,” as used herein, refers to a mixturecontaining equimolar quantities of two enantiomers of a compound. Suchmixtures exhibit no optical activity (i.e., they do not rotate a planeof polarized light).

Percent enantiomeric excess (ee) is defined as the absolute differencebetween the mole fraction of each enantiomer multiplied by 100% and canbe represented by the following equation:

${{e\; e} = {{\frac{R - S}{R + S}} \times 100\%}},$where R and S represent the respective fractions of each enantiomer in amixture, such that R+S=1. When a single enantiomer is named or depictedby structure, the depicted or named enantiomer is present in an ee of atleast or about 50%, about 60%, about 70%, about 80%, about 90%, about95%, about 98%, about 99% or about 99.9%.

Percent diastereomeric excess (de) is defined as the absolute differencebetween the mole fraction of each diastereomer multiplied by 100% andcan be represented by the following equation:

${{d\; e} = {{\frac{{D\; 1} - \left( {{D\; 2} + {D\; 3} + {D\; 4\mspace{11mu}\ldots}}\mspace{11mu} \right)}{{D\; 1} + \left( {{D\; 2} + {D\; 3} + {D\; 4\mspace{11mu}\ldots}}\mspace{11mu} \right)}} \times 100\%}},$where D1 and (D2+D3+D4 . . . ) represent the respective fractions ofeach diastereomer in a mixture, such that D1+(D2+D3+D4 . . . )=1. When asingle diastereomer is named or depicted by structure, the depicted ornamed diastereomer is present in a de of at least or about 50%, about60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99% orabout 99.9%.

When a disclosed compound is named or depicted by structure withoutindicating the stereochemistry, and the compound has one chiral center,it is to be understood that the name or structure encompasses oneenantiomer of the compound substantially separated from thecorresponding optical isomer, a racemic mixture of the compound andmixtures enriched in one enantiomer relative to its correspondingoptical isomer.

When a disclosed compound is named or depicted by structure withoutindicating the stereochemistry and has two or more chiral centers, it isto be understood that the name or structure encompasses a diastereomersubstantially separated from other diastereomers, a pair ofdiastereomers substantially separated from other diastereomeric pairs,mixtures of diastereomers, mixtures of diastereomeric pairs, mixtures ofdiastereomers in which one diastereomer is enriched relative to theother diastereomer(s) and mixtures of diastereomeric pairs in which onediastereomeric pair is enriched relative to the other diastereomericpair(s).

“(R)-Lipoyl” refers to a compound containing a lipoyl moiety, whereinthe stereocenter in the lipoyl moiety is in the (R) configuration. An(R)-lipoyl moiety is pictured below:

An example of an (R)-lipoyl compound is shown below:

“(S)-Lipoyl” refers to a compound containing a lipoyl moiety, whereinthe stereocenter in the lipoyl moiety is in the (S) configuration. An(S)-lipoyl moiety is pictured below:

An example of an (S)-lipoyl compound is shown below:

“Alkyl” means a saturated aliphatic branched or straight-chainmonovalent hydrocarbon radical having the specified number of carbonatoms. Thus, “(C₁-C₆)alkyl” means a radical having from 1-6 carbon atomsin a linear or branched arrangement. “(C₁-C₆)alkyl” includes methyl,ethyl, propyl, i-propyl, butyl, i-butyl, t-butyl, sec-butyl, pentyl andhexyl. Typically, alkyl has 1 to 20, 1 to 15, 1 to 10, 1 to 5 or 1 to 3carbon atoms.

One or more hydrogen atoms of an alkyl group can be replaced with asubstituent group. Suitable substituent groups include hydroxy, thio,halo, halo(C₁-C₃)alkyl, (C₁-C₃)alkoxy and thio(C₁-C₃)alkyl. Preferredalkyl substituent groups include hydroxy and halo. An alkyl can also besubstituted with one or more acidic substituents selected from the groupconsisting of —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H, —OPO₃H₂, —B(OH)₂ and —NHOH.

The term “alkoxy” means —O-alkyl, where alkyl is as defined above.

The term “halogen” means F, Cl, Br or I.

The term “aryl” means a carbocyclic aromatic ring. “(C₆-C₁₄)aryl”includes phenyl, napthyl, indenyl, and anthracenyl. Typically, aryl has6 to 20, 6 to 14, 6 to 10, 6 to 9, or 6 carbon atoms.

As used herein, “substantially separated” or “substantially pure” meansthat the ee or de of the depicted or named compound is at least about50%. For example, “substantially separated” or “substantially pure” canmean the ee or de of the depicted or named enantiomer is at least orabout 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about98%, about 99% or about 99.9%. In one embodiment, substantiallyseparated or substantially pure means that the ee or de of the depictedor named compound is at least or about 75%. In a specific embodiment,substantially separated means that the ee or de of the depicted or namedcompound is at least or about 90%. In a more specific embodiment,substantially separated means that the ee or de of the depicted or namedcompound is at least or about 95%. In yet a more specific embodiment,substantially separated means that the ee or de of the depicted or namedcompound is at least or about 99%. In another specific embodiment,substantially separated means that the ee or de of the depicted or namedcompound is at least or about 99.9%.

As used herein, the term “amino acid” means a molecule containing anamine group, a carboxylic acid group and a side chain which variesbetween different amino acids and includes both naturally-occurringamino acids and non-naturally-occurring amino acids. In one embodiment,“amino acid” is used to refer to naturally-occurring amino acids.

As used herein, the term “naturally-occurring amino acid” means acompound represented by the formula NH₂—CHR—COOH, wherein R is the sidechain of a naturally-occurring amino acid such as an amino acid listedor named in the Table below. “Naturally-occurring amino acid” includesboth the D- and L-configuration. When an amino acid is named or depictedby structure without indicating the stereochemistry and has at least onechiral center, it is to be understood that the name or structureencompasses a single enantiomer or diastereomer substantially separatedfrom the other enantiomer or diastereomer, in which the one enantiomeror diastereomer is enriched relative to the other enantiomer ordiastereomer(s), a racemic or diastereomeric mixture of the enantiomeror diastereomer(s) and mixtures enriched in one enantiomer ordiastereomer relative to its corresponding optical isomer or otherdiastereomer(s).

Table of Common Naturally Occurring Amino Acids Amino acid Three lettercode One letter code Non-polar; alanine Ala A neutral at isoleucine IleI pH 7.4 leucine Leu L methionine Met M phenylalanine Phe F proline ProP tryptophan Trp W valine Val V Polar, asparagine Asn N unchargedcysteine Cys C at pH 7.0 glycine Gly G glutamine Gln Q serine Ser Sthreonine Thr T tyrosine Tyr Y Polar; glutamic acid Glu E charged atarginine Arg R pH 7 aspartic acid Asp D histidine His H lysine Lys K

“Non-natural amino acid” means an amino acid for which there is nonucleic acid codon. Examples of non-natural amino acids include naturalα-amino acids with non-natural side chains (e.g., entry 6 and 7 in Table2); β-amino acids (e.g., β-alanine); γ-amino acids (e.g.,γ-aminobutryric acid).

As used herein, an “effective amount” is an amount sufficient to achievea desired therapeutic or prophylactic effect in a subject in needthereof under the conditions of administration, such as, for example, anamount sufficient to inhibit (e.g., prevent, delay) ischemia andischemia-reperfusion injury in a subject (e.g., by inhibiting cell deathof one or more affected cells in the subject). The effectiveness of atherapy can be determined by suitable methods known by those of skill inthe art. An effective amount includes any amount of a compound (e.g., acompound of Structural Formula (I) and/or (Ia)) which prevents the onsetof, alleviates the symptoms of, or stops the progression of a disorderor disease being treated (e.g., ischemia or ischemia-reperfusion injury)in a subject.

The term “treating” is defined as administering to a subject in needthereof an effective amount of a compound (e.g., of Structural Formula(I) and/or (Ia), or a pharmaceutically acceptable salt or prodrugthereof) that is sufficient to prevent the onset of, alleviate thesymptoms of, or stop the progression of a disorder or disease beingtreated.

The term “subject,” as used herein, refers to a mammal. In a preferredembodiment, the subject is a human.

Compounds of the Invention

The present invention relates in one embodiment to a compoundrepresented by Structural Formula (I) and/or (Ia).

R is (C₁-C₁₈)alkyl, (C₆-C₁₈)aryl or (C₆-C₁₈)aryl(C₁-C₁₈)alkyl and issubstituted with at least one acidic substituent selected from the groupconsisting of —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H, —OPO₃H₂, —B(OH)₂ and —NHOH,wherein the aryl of the (C₆-C₁₈)aryl or (C₆-C₁₈)aryl(C₁-C₁₈)alkyl isoptionally further substituted with one or more substituents selectedfrom the group consisting of hydroxy, halo, (C₁-C₃)alkyl,halo(C₁-C₃)alkyl, cyano, nitro, (C₁-C₃)alkoxy and thio(C₁-C₃)alkyl.

In one embodiment, R is (C₁-C₁₈)alkyl. In another embodiment, R is(C₁-C₃)alkyl. In a further embodiment, R is (C₃)alkyl. In a furtherembodiment, R is (C₂)alkyl. Alternatively, R is (C₁)alkyl.

In another embodiment, R is (C₆-C₁₈)aryl. In a further embodiment, R is(C₆)aryl.

In another embodiment, R is (C₆-C₁₈)aryl(C₁-C₁₈)alkyl. In a furtherembodiment, R is (C₆)aryl(C₁-C₃)alkyl. Alternatively, R is(C₆)aryl(C₁-C₂)alkyl.

In another embodiment, R is (C₆)aryl(C₂)alkyl. In a further embodiment,R is (C₆)aryl(C₁)alkyl.

The at least one acidic substituent is selected from the groupconsisting of —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H, —OPO₃H₂, —B(OH)₂ and —NHOH.In one embodiment, the at least one acidic substituent is selected fromthe group consisting of —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H and —OPO₃H₂.

R is substituted with at least one acidic substituent selected from thegroup consisting of —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H, —OPO₃H₂, —B(OH)₂ and—NHOH. In one embodiment, R is substituted with one, two or three acidicsubstituents. In a further embodiment, R is substituted with one or twoacidic substituents.

Aryl is optionally further substituted with one or more substituentsselected from the group consisting of hydroxy, halo, (C₁-C₃)alkyl,halo(C₁-C₃)alkyl, cyano, nitro, (C₁-C₃)alkoxy and thio(C₁-C₃)alkyl. Inone embodiment, aryl is further substituted with one, two or threesubstituents. In another embodiment, aryl is substituted with onesubstituent. Alternatively, aryl is unsubstituted. In a furtherembodiment, aryl is further substituted with one or more substituentsselected from the group consisting of hydroxyl and halo.

R′ is hydrogen or (C₁-C₁₈)alkyl, wherein said (C₁-C₁₈)alkyl isoptionally substituted with one or more acidic substituents selectedfrom the group consisting of —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H, —OPO₃H₂,—B(OH)₂ and —NHOH. In one embodiment, R′ is hydrogen.

In one embodiment, R′ is (C₁-C₁₈)alkyl. In another embodiment, R′ is(C₁-C₃)alkyl. In a further embodiment, R′ is (C₃)alkyl. In a furtherembodiment, R′ is (C₂)alkyl. Alternatively, is (C₁)alkyl.

R′ is substituted with at least one acidic substituent selected from thegroup consisting of —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H, —OPO₃H₂, —B(OH)₂ and—NHOH. In one embodiment, R′ is substituted with one, two or threeacidic substituents. In another embodiment, R′ is substituted with oneor two acidic substituents. In a further embodiment, R′ is substitutedwith one acidic substituent. Alternatively, R′ is unsubstituted.

X is absent or an amino acid, wherein the amino acid is oriented to forman amide linkage with

For example, the moiety in N-lipoyl-glutamylalanine is oriented as shownin Structural Formula below:

In one embodiment, X is absent. Alternatively, X is an amino acid. In afurther embodiment, X is a naturally-occurring amino acid. In yet afurther embodiment, X is aspartic acid, tyrosine, glutamic acid oralanine.

The compounds of Structural Formulas (I) and/or (Ia) are notN—(R)-lipoyl-glutamylalanine, N-lipoyl-aspartylglycine,N-lipoyl-glutamylglycine, N—(R)-lipoyl-aminoethylphosphonic acid, or(R)-5-(5-(1,2-dithiolan-3-yl)pentanamido)-2-hydroxybenzoic acid.

The compounds of Structural Formulas (I) and/or (Ia) are notN-lipoyl-glutamylalanine, N-lipoyl-aspartylglycine,N-lipoyl-glutamylglycine, N-lipoyl-glutamine, N-lipoyl-glycine or5-(5-(1,2-dithiolan-3-yl)pentanamido)-2-hydroxybenzoic acid.

In addition, in specific embodiments, the compound of StructuralFormulas (I) and/or (Ia) is not N-lipoyl-aspartylalanine.

In a 1^(st) specific embodiment, the compound is represented byStructural Formula (I) and/or (Ia), wherein the values and alternativevalues for the variables are as described above.

In a first aspect of the 1^(st) specific embodiment of the presentinvention, the (R)-lipoyl stereoisomer of a compound represented byStructural Formulas (I) or (Ia), or a pharmaceutically acceptable saltor prodrug thereof, is substantially separated from the (S)-lipoylstereoisomer(s) or a pharmaceutically acceptable salt or prodrugthereof. Values and alternative values for the remainder of thevariables are as described above for Structural Formulas (I) or (Ia) orin the 1^(st) specific embodiment.

In a second aspect of the 1^(st) specific embodiment of the presentinvention, R′ is H. Values and alternative values for the remainder ofthe variables are as described above for Structural Formulas (I) or (Ia)or in the 1^(st) specific embodiment, or first aspect thereof.

In a third aspect of the 1^(st) specific embodiment of the presentinvention, R′ is H and X is a naturally-occurring amino acid. Values andalternative values for the remainder of the variables are as describedabove for Structural Formulas (I) or (Ia) or in the 1^(st) specificembodiment, or first or second aspect thereof.

In a fourth aspect of the 1^(st) specific embodiment of the presentinvention, R and R′ are each (C₁-C₃)alkyl substituted with one or twoacidic substituents each independently selected from —CO₂H, —SO₃H,—PO₃H₂, —OSO₃H and —OPO₃H₂. Values and alternative values for theremainder of the variables are as described above for StructuralFormulas (I) or (Ia) or in the 1^(st) specific embodiment, or first tothird aspects thereof.

In a fifth aspect of the 1^(st) specific embodiment of the presentinvention, R′ is H and X is absent. Values and alternative values forthe remainder of the variables are as described above for StructuralFormulas (I) or (Ia) or in the 1^(st) specific embodiment, or first tofourth aspects thereof.

In a sixth aspect of the 1^(st) specific embodiment of the presentinvention, R is (C₁-C₃)alkyl substituted with one or two acidicsubstituents each independently selected from —CO₂H, —SO₃H, —PO₃H₂,—OSO₃H and —OPO₃H₂. Values and alternative values for the remainder ofthe variables are as described above for Structural Formulas (I) or (Ia)or in the 1^(st) specific embodiment, or first to fifth aspects thereof.

In a seventh aspect of the 1^(st) specific embodiment of the presentinvention, R is (C₆)aryl(C₁-C₃)alkyl substituted with one or two acidicsubstituents each independently selected from —CO₂H, —SO₃H, —PO₃H₂,—OSO₃H and —OPO₃H₂. Values and alternative values for the remainder ofthe variables are as described above for Structural Formulas (I) or (Ia)or in the 1^(st) specific embodiment, or first to sixth aspects thereof.

In an eighth aspect of the 1^(st) specific embodiment of the presentinvention, R is (C₂)alkyl substituted with one or two acidicsubstituents each independently selected from —CO₂H, —SO₃H, —PO₃H₂,—OSO₃H and —OPO₃H₂. Values and alternative values for the remainder ofthe variables are as described above for Structural Formulas (I) or (Ia)or in the 1^(st) specific embodiment, or first to seventh aspectsthereof.

In a ninth aspect of the 1^(st) specific embodiment of the presentinvention, R is (C₆)aryl substituted with one acidic substituentselected from —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H and —OPO₃H₂. Values andalternative values for the remainder of the variables are as describedabove for Structural Formulas (I) or (Ia) or in the 1^(st) specificembodiment, or first to eighth aspects thereof.

In a tenth aspect of the 1^(st) specific embodiment of the presentinvention, the compound represented by Structural Formulas (I) and/or(Ia) is not N-lipoyl-glutamylalanine, N-lipoyl-aspartylglycine,N-lipoyl-glutamylglycine or5-(5-(1,2-dithiolan-3-yl)pentanamido)-2-hydroxybenzoic acid. Values andalternative values for the remainder of the variables are as describedabove for Structural Formulas (I) or (Ia) or in the 1^(st) specificembodiment, or first to ninth aspects thereof.

In an eleventh aspect of the 1^(st) specific embodiment of the presentinvention, the compound represented by Structural Formulas (I) and/or(Ia) is not N-lipoyl-glutamylalanine, N-lipoyl-aspartylglycine,N-lipoyl-glutamylglycine, N-lipoyl-glutamic acid, N-lipoyl-asparticacid, N-lipoyl-glycine or5-(5-(1,2-dithiolan-3-yl)pentanamido)-2-hydroxybenzoic acid. Values andalternative values for the remainder of the variables are as describedabove for Structural Formulas (I) or (Ia) or in the 1^(st) specificembodiment, or first to tenth aspects thereof.

In a twelfth aspect of the 1^(st) specific embodiment of the presentinvention, the compound of Structural Formula (I) is notN—(R)-lipoyl-glutamylalanine, N—(R)-lipoyl-aspartylglycine,N—(R)-lipoyl-aminoethylphosphonic acid, or(R)-5-(5-(1,2-dithiolan-3-yl)pentanamido)-2-hydroxybenzoic acid. Valuesand alternative values for the remainder of the variables are asdescribed above for Structural Formulas (I) or (Ia) or in the 1^(st)first specific embodiment, or first to eleventh aspects thereof.

In a thirteenth aspect of the first specific embodiment, the compound isrepresented by Structural Formula (I), wherein the values andalternative values are as described above for Structural Formulas (I) or(Ia) or in the 1^(st) specific embodiment, or first to twelfth aspectsthereof.

In a fourteenth aspect of the 1^(st) specific embodiment, the compoundis represented by Structural Formula (Ia), wherein the values andalternative values are as described above for Structural Formulas (I) or(Ia) or in the 1^(st) specific embodiment, or first to thirteenthaspects thereof.

In a 2^(nd) specific embodiment, the compound is represented by one ofthe following structural formulas:

Values and alternative values for the remainder of the variables are asdescribed above for Structural Formulas (I) or (Ia) or in the 1^(st)specific embodiment, or aspects thereof.

In a first aspect of the 2^(nd) specific embodiment of the presentinvention, the (R)-lipoyl stereoisomer of a compound represented byStructural Formulas (I) or (Ia), or a pharmaceutically acceptable saltor prodrug thereof, is substantially separated from the (S)-lipoylstereoisomer(s) or a pharmaceutically acceptable salt or prodrugthereof. Values and alternative values for the remainder of thevariables are as described above for Structural Formula (Ia), in the1^(st) specific embodiment, or aspects thereof, or in the 2^(nd)specific embodiment.

The invention also relates to pharmaceutically acceptable salts of thedisclosed compounds of the present invention. The term “pharmaceuticallyacceptable salts” embraces salts commonly used to form alkali metalsalts and to form addition salts of free bases. The nature of the saltis not critical, provided that it is pharmaceutically acceptable.

The pharmaceutically acceptable salts of the compounds of the presentinvention include base addition salts. Suitable pharmaceuticallyacceptable base addition salts of compounds of the present inventioninclude, but are not limited to, metallic salts made from aluminum,calcium, lithium, magnesium, potassium, sodium and zinc or organic saltsmade from N,N′-dibenzylethylene-diamine, chloroprocaine, choline,diethanolamine, ethylenediamine, N-methylglucamine, lysine and procaine.All of these salts may be prepared by conventional means from acorresponding compound of the present invention by treating, forexample, a compound of Tables 1-5 with the appropriate acid or base.

In one embodiment, the pharmaceutically acceptable salt comprises amonovalent or divalent cation. As used herein, “cation” refers to anatom or molecule that has a positive charge. A cation can be, forexample, a metal or an amine. In a particular embodiment, the cation isa metal cation, such as a sodium cation.

As used herein, “amine salt” relates to a cation containing a protonatedamino group. Amine salts include amino acid salts, such as lysine salts.In another embodiment, the cation is an amine and the pharmaceuticallyacceptable salt is an amine salt. In a particular embodiment, thepharmaceutically acceptable salt comprises lysine.

Salts can be chiral. When a disclosed salt has at least one chiralcenter and is named or depicted by structure without indicating thestereochemistry, it is to be understood that the name or structureencompasses one stereoisomer or enantiomer of the compound free from thecorresponding stereoisomer(s) or enantiomer, a racemic mixture of thecompound, or mixtures enriched in one stereoisomer or enantiomerrelative to its corresponding stereoisomer(s) or enantiomer.

The invention also relates to pharmaceutically acceptable prodrugs ofthe disclosed compounds of the present invention.

In one embodiment, the invention relates to the compounds of StructuralFormulas (I) and/or (Ia), wherein the hydrogen of each acidicfunctionality (e.g., —COOH, —SO₃H, —OSO₃H, —PO(OH)₂, —OPO(OH)₂) isoptionally and independently replaced with a hydrolyzable group. Theinvention also encompasses pharmaceutically acceptable salts of thecompounds including said hydrolyzable groups.

As used herein, the term “hydrolyzable group” refers to a moiety that,when present in a molecule of the invention, yields a carboxylic acid,or salt thereof, upon hydrolysis. Hydrolysis can occur, for example,spontaneously under acidic or basic conditions in a physiologicalenvironment (e.g., blood, metabolically active tissues, for example,liver, kidney, lung, brain), or can be catalyzed by an enzyme(s), (e.g.,esterase, peptidases, hydrolases, oxidases, dehydrogenases, lyases orligases). A hydrolyzable group can confer upon a compound of theinvention advantageous properties in vivo, such as improved watersolubility, improved circulating half-life in the blood, improveduptake, improved duration of action, or improved onset of action.

In one embodiment, the hydrolyzable group does not destroy thebiological activity of the compound. In an alternative embodiment, acompound with a hydrolyzable group can be biologically inactive, but canbe converted in vivo to a biologically active compound.

Compounds of the invention that include hydrolyzable groups may act asprodrugs. As used herein, the term “prodrug” means a compound that canbe hydrolyzed, oxidized, metabolized or otherwise react under biologicalconditions to provide a compound of the invention. Prodrugs may becomeactive upon such reaction under biological conditions, or they may haveactivity in their unreacted forms. A prodrug may undergo reducedmetabolism under physiological conditions (e.g., due to the presence ofa hydrolyzable group), thereby resulting in improved circulatinghalf-life of the prodrug (e.g., in the blood). Prodrugs can typically beprepared using well-known methods, such as those described by Burger'sMedicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (ManfredE. Wolff ed., 5^(th) Ed).

In one embodiment, the hydrolyzable group is selected from the groupconsisting of (C₁-C₁₀)alkyl, (C₂-C₁₀)alkenyl, (C₂-C₁₀)alkynyl,(C₁-C₁₀)alkoxy(C₁-C₁₀)alkyl, (C₁-C₁₀)alkoxy(C₁-C₁₀)alkoxy(C₁-C₁₀)alkyl,aryl and aryl(C₁-C₁₀)alkyl, wherein each is optionally substituted with1 to 3 substituents selected from the group consisting of halo, nitro,cyano, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, amino,(C₁-C₆)alkylamino, di(C₁-C₆)alkylamino, (C₁-C₆)alkyl, halo(C₁-C₆)alkyl,(C₁-C₆)alkoxy, halo(C₁-C₆)alkoxy, morpholino, phenyl, and benzyl.

In another embodiment, the hydrolyzable group is selected from the groupconsisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,isobutyl, tert-butyl, pentyl, hexyl, heptyl, allyl, ethoxymethyl,methoxyethyl, methoxyethoxymethyl, methoxyethoxyethyl, benzyl,pentafluorophenyl, 2-N-(morpholino)ethyl, dimethylaminoethyl andpara-methoxybenzyl.

Methods

In another embodiment, the invention relates to a method for treating anischemia or ischemia-reperfusion injury in a subject in need thereof,comprising administering to the subject an effective amount of one ormore compounds of Structural Formulas (I) and/or (Ia), or apharmaceutically acceptable salt or prodrug thereof, provided that thecompounds of Structural Formula (I) are notN—(R)-lipoyl-glutamylalanine, N—(R)-lipoyl-aminoethylphosphonic acid, or(R)-5-(5-(1,2-dithiolan-3-yl)pentanamido)-2-hydroxybenzoic acid and thecompounds of Structural Formula (Ia) are not N-lipoyl-glutamylalanine,N-lipoyl-aspartylglycine, N-lipoyl-glutamylglycine or5-(5-(1,2-dithiolan-3-yl)pentanamido)-2-hydroxybenzoic acid. In someembodiments, the pharmaceutically acceptable salt is a lysine salt. Inone embodiment, the salt is an L-lysine salt. In a particularembodiment, the ischemia or ischemia-reperfusion injury is a myocardialischemia or ischemia-reperfusion injury. In another embodiment, thecompound is administered as a composition comprising one or morecompounds of the invention.

As used herein, the “injury resulting from ischemia,” “injury caused byischemia” and “ischemic injury” refer to an injury to a cell, tissue ororgan caused by ischemia, or an insufficient supply of blood (e.g., dueto a blocked artery), and thus oxygen, resulting in damage ordysfunction of the tissue or organ (Piper, H. M., Abdallah, C., Schafer,C., Annals of Thoracic Surgery 2003, 75:644; Yellon, D. M., Hausenloy,D. J., New England Journal of Medicine 2007, 357:1121). Injuries thatresult from ischemia can affect various tissues and organs. Suchinjuries may be treated by the compounds and methods of the invention,including, for example, injuries caused by cardiovascular ischemia,cerebrovascular ischemia, renal ischemia, hepatic ischemia, ischemiccardiomyopathy, cutaneous ischemia, bowel ischemia, intestinal ischemia,gastric ischemia, pulmonary ischemia, pancreatic ischemia, skeletalmuscle ischemia, abdominal muscle ischemia, limb ischemia, ischemiccolitis, mesenteric ischemia and silent ischemia. Thus, an injuryresulting from ischemia can affect, for example, a heart, kidney, liver,brain, muscle, intestine, stomach, lung or skin.

In a particular embodiment, the injury resulting from ischemia is theresult of a myocardial ischemia. An injury resulting from a myocardialischemia can result from, for example, a myocardial infarction (e.g., anacute myocardial infarction) in a subject.

In another embodiment, the injury resulting from ischemia is an injuryresulting from cerebral ischemia (e.g., a stroke) in a subject.

In another embodiment, the injury resulting from ischemia is an injuryresulting from renal ischemia. An injury resulting from a renal ischemiacan result from, for example, a deficiency of blood in one or bothkidneys, or nephrons, usually due to functional constriction or actualobstruction of a blood vessel (e.g., an acute renal infarction) in asubject.

In another embodiment, the injury resulting from ischemia is anischemia-reperfusion injury. As used herein, the term“ischemia-reperfusion injury” refers to an injury resulting from therestoration of blood flow to an area of a tissue or organ that hadpreviously experienced deficient blood flow due to an ischemic event.Oxidative stresses associated with reperfusion may cause damage to theaffected tissues or organs. Ischemia-reperfusion injury is characterizedbiochemically by a depletion of oxygen during an ischemic event followedby reoxygenation and the concomitant generation of reactive oxygenspecies during reperfusion (Piper, H. M., Abdallah, C., Schafer, C.,Annals of Thoracic Surgery 2003, 75:644; Yellon, D. M., Hausenloy, D.J., New England Journal of Medicine 2007, 357:1121).

An ischemia-reperfusion injury can be caused, for example, by a naturalevent (e.g., restoration of blood flow following a myocardialinfarction), a trauma, or by one or more surgical procedures or othertherapeutic interventions that restore blood flow to a tissue or organthat has been subjected to a diminished supply of blood. Such surgicalprocedures include, for example, coronary artery bypass graft surgery,coronary angioplasty, organ transplant surgery and the like. In aparticular embodiment, the compounds and methods of the invention areuseful for treating peri-operative cardiac damage caused by an ischemiaor ischemia-reperfusion injury.

For the treatment of ischemic and ischemia-reperfusion injuries causedby therapeutic interventions, such as surgical procedures, it ispreferable that a compound of the invention is administered to a subjectundergoing treatment prior to the therapeutic intervention (e.g.,cardiac surgery, organ transplant). For example, a compound of theinvention can be administered to a subject undergoing treatment, e.g.,about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5hours, about 12 hours, about 24 hours, or about 48 hours prior to thetherapeutic intervention. A compound of the invention can also beadministered to a subject undergoing treatment, for example, about 5minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30minutes or about 45 minutes prior to the therapeutic intervention.

Alternatively, or in addition, a compound of the invention can beadministered to a subject undergoing treatment at the time of, orduring, the therapeutic intervention. For example, the compound can beadministered one or more times during the course of a therapeuticintervention in intervals (e.g., 15 minute intervals). Alternatively, acompound can be administered continuously throughout the duration of atherapeutic intervention.

Furthermore, a compound of the invention can be administered to asubject undergoing treatment after a therapeutic intervention. Forexample, a compound of the invention can be administered to a subjectundergoing treatment, e.g., about 1 hour, about 2 hours, about 3 hours,about 4 hours, about 5 hours, about 12 hours, about 24 hours, or about48 hours after the therapeutic intervention. A compound of the inventioncan also be administered to a subject undergoing treatment, for example,about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes,about 30 minutes or about 45 minutes after the therapeutic intervention.

A compound of the invention can also be used to inhibit an ischemia orischemia-reperfusion injury to a cell, tissue or organ, ex vivo, priorto a therapeutic intervention (e.g., a tissue employed in a graftprocedure, an organ employed in an organ transplant surgery). Forexample, prior to transplant of an organ into a host individual (e.g.,during storage or transport of the organ in a sterile environment), theorgan can be contacted with a compound of the invention (e.g., bathed ina solution comprising a compound of the invention) to inhibit ischemiaor ischemia-reperfusion injury.

As described herein, conditions resulting from ischemia, and injuriescaused by ischemia or ischemia-reperfusion, can induce cell death (e.g.,apoptotic cell death) in an affected cell, tissue or organ, leading todamage and dysfunction. Accordingly, the compounds of the invention alsohave utility in methods of inhibiting cell death in a cell, a tissue oran organ (e.g., a transplant tissue or organ or a cell, tissue or organin a subject), wherein the cell, tissue or organ has experienced anischemia or other condition or disorder that results in excessive orunwanted cell death. The methods comprise contacting the cells, tissue,or organ in need thereof with, or administering to a subject in needthereof, an effective amount of one or more compounds of StructuralFormulas (I) and/or (Ia), or a pharmaceutically acceptable salt orprodrug thereof.

In one embodiment, the invention relates to a method of inhibiting celldeath (e.g., apoptotic cell death) in a subject, comprisingadministering to the subject an effective amount of a compoundrepresented by Structural Formula (I) or (Ia), or a pharmaceuticallyacceptable salt or prodrug thereof.

Methods of assessing cell death are well known in the art. For example,microscopic analysis (e.g., light microscopy, electron microscopy,confocal microscopy, laser-scanning microscopy) for visualizing celldeath (e.g., by detecting morphological changes associated with celldeath, such as chromatin condensation and cytoplasmic shrinking) istypically employed to study cell death.

The study of DNA fragmentation in agarose gels is also considered to beindicative of apoptotic cell death. A number of techniques takeadvantage of DNA fragmentation for labeling the fragments and thus forquantifying the proportion of apoptotic cells. Each DNA fragment has a3′-OH terminal portion. This terminal fragment can be labeled in variousways (for instance, with the help of a modified terminaldeoxynucleotidyl transferase), so that the labeling rate is proportionalto the degree of DNA fragmentation.

In particular, TdT-mediated dUTP Nick-End Labeling, or TUNEL, is atechnique for detecting fragmented DNA, which occurs near the final stepin the apoptotic process. Fragmented DNA of apoptotic cells canincorporate fluorescein-dUTP at 3′-OH at DNA ends using the enzymeTerminal Deoxynucleotidyl Transferase (TdT), which forms a polymerictail using the principle of the TUNEL assay. The labeled DNA can then bevisualized directly by fluorescence microscopy or quantitated by flowcytometry.

Some current techniques take advantage of the changes in membranephospholipids that occur early in apoptotic cells. The negativelycharged membrane phospholipids exposed to the external environment bythe apoptotic cell are labeled with fluorochrome-conjugated molecules,and the percentage of fluorescent cells can be easily quantified.

Apoptosis can also be detected using fluorescently-conjugated Annexin V.Annexin V is an anticoagulant protein that preferentially bindsnegatively charged phospholipids. An early step in the apoptotic processis disruption of membrane phospholipid asymmetry, exposingphosphatidylserine (PS) on the outer leaflet of the cytoplasmicmembrane. Fluorescently conjugated Annexin V can be used to detect thisexternalization of phosphatidylserine on intact living cells. Propidiumiodide is often combined as a second fluorochrome to detect necroticcells. Induction of apoptosis leads to procaspase-3 proteolytic cleavageto generate an active 18 kDa caspase-3 fragment which then targets keymodulators of the apoptotic pathway including poly-ADP-ribose polymeraseand other caspases, for cleavage. Assays for detecting other activecaspases in apoptotic cells are known in the art (e.g., Caspase-Glo®Assays, Promega).

Apoptotic cells can also be detected using the active 18 kDa caspase-3fragment as a marker. Induction of apoptosis leads to procaspase-3proteolytic cleavage to generate an active 18 kDa caspase-3 fragmentwhich then targets key modulators of the apoptotic pathway, includingpoly-ADP-ribose polymerase and other caspases, for cleavage. Severalantibodies that recognize only the active 18 kDa fragment are availablefrom commercial suppliers (e.g., BD Biosciences, Chemicon, CellSignaling Technology, Trevigen).

In addition, flow cytometry assays can be employed to monitor andquantify nuclear changes associated with apoptotic cells.

Conditions associated with unwanted and/or excess cell death that aretreatable by the compounds and methods of the invention include, but arenot limited to, neurodegenerative diseases associated with excess celldeath (e.g., Parkinson's Disease, Alzheimer's Disease, amyotrophiclateral sclerosis, retinitis pigmentosa, epilepsy), haematologicdiseases associated with excess cell death (e.g., aplastic anaemia,myelodysplastic syndrome, T CD4+ lymphocytopenia, G6PD deficiency),tissue damage associated with excess apopotosis (e.g., myocardialinfarction, cerebrovascular accident, ischemic renal damage, polycystickidney disease), AIDS, and preeclampsia.

The invention also relates to compositions comprising a pharmaceuticalacceptable carrier or diluent and one or more of the disclosedcompounds, or a pharmaceutically acceptable salt or prodrug thereof. Thecompositions disclosed herein are prepared in accordance with standardprocedures and are administered at dosages that are selected to reduce,prevent, eliminate, or to slow or halt the progression of, the conditionbeing treated. See, e.g., Remington's Pharmaceutical Sciences, 17^(th)ed., Remington, J. P., Easton, Pa., Mack Publishing Company, 2005, andGoodman and Gilman's The Pharmaceutical Basis of Therapeutics, 12^(th)ed., Brunton, L. et. als., eds., New York, McGraw-Hill, 2010, thecontents of which are incorporated herein by reference, for a generaldescription of the methods for administering various agents for humantherapy. The compositions of the invention can be delivered usingcontrolled or sustained-release delivery systems (e.g., capsules,bioerodable matrices). Exemplary delayed-release delivery systems fordrug delivery that would be suitable for administration of thecompositions of the present invention are described in U.S. Pat. No.5,990,092 (issued to Walsh); U.S. Pat. No. 5,039,660 (issued toLeonard); U.S. Pat. No. 4,452,775 (issued to Kent); and U.S. Pat. No.3,854,480 (issued to Zaffaroni).

The compositions of the present invention comprise one or more compoundsof Structural Formulas (I) and/or (Ia), or a pharmaceutically acceptablesalt or prodrug thereof, in association with one or more nontoxic,pharmaceutically acceptable carriers and/or diluents and/or adjuvantsand/or excipients, collectively referred to herein as “carrier”materials, and optionally, other active ingredients. The compositionsmay contain from about 0.01% to about 99% by weight of the activeingredient, depending on the method of administration.

For preparing compositions from the compounds of the present invention,pharmaceutically acceptable carriers can either be solid or liquid.Solid form preparations include powders, tablets, pills, capsules,cachets, suppositories, and dispersible granules. For example, thecompounds of the present invention may be in powder form forreconstitution at the time of delivery. A solid carrier can be one ormore substances which may also act as diluents, flavoring agents,solubilizers, lubricants, suspending agents, binders, preservatives,tablet disintegrating agents, or an encapsulating material. In powders,the carrier is a finely divided solid which is in a mixture with thefinely divided active ingredient.

In tablets, the active ingredient is mixed with the carrier having thenecessary binding properties in suitable proportions and compacted inthe shape and size desired.

The powders and tablets preferably contain from about one to aboutseventy percent of the active ingredient. Suitable carriers aremagnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin,dextrin, starch, gelatin, tragacanth, methylcellulose, sodiumcaboxymethylcellulose, a low-melting wax, cocoa butter, and the like.Tablets, powders, cachets, lozenges, fast-melt strips, capsules andpills can be used as solid dosage forms containing the active ingredientsuitable for oral administration.

Liquid form preparations include solutions, suspensions, retentionenemas, and emulsions, for example, water or water propylene glycolsolutions. For parenteral injection, liquid preparations can beformulated in solution in aqueous polyethylene glycol.

Aqueous solutions suitable for oral administration can be prepared bydissolving the active ingredient in water and adding suitable colorants,flavors, stabilizing agents, and thickening agents as desired. Aqueoussuspensions for oral administration can be prepared by dispersing thefinely divided active ingredient in water with viscous material, such asnatural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, and other well-known suspending agents.

Alternatively, the compounds or compositions of the present inventioncan be in powder form for reconstitution at the time of delivery.

The composition is preferably in unit dosage form. In such form, thecomposition is subdivided into unit doses containing appropriatequantities of the active ingredient. The unit dosage form can be apackaged preparation, the package containing discrete quantities of, forexample, tablets, powders, and capsules in vials or ampules. Also, theunit dosage form can be a tablet, cachet, capsule, or lozenge itself, orit can be the appropriate amount of any of these in packaged form. Thequantity of active ingredient in a unit dose preparation may be variedor adjusted from about 0.1 mg to about 1000 mg, preferably from about0.1 mg to about 100 mg (e.g., for intravenous administration) or fromabout 1.0 mg to about 1000 mg (e.g., for oral administration). Thedosages, however, may be varied depending upon the requirements of thesubject, the severity of the condition being treated, the compound andthe route of administration being employed. Determination of the properdosage for a particular situation is within the skill in the art. In oneembodiment, the dosage is from about 0.01 mg/kg to about 100 mg/kg.

In general, the methods for delivering the disclosed compounds andpharmaceutical compositions of the invention in vivo utilizeart-recognized protocols for delivering the agent with the onlysubstantial procedural modification being the substitution of thecompounds represented by any one of the disclosed compounds for thedrugs in the art-recognized protocols.

The compounds of the present invention may be administered by any route,preferably in the form of a composition adapted to such a route, andwould be dependent on the condition being treated. The compounds andcompositions may, for example, be administered intravascularly,intramuscularly, subcutaneously, intraperitoneally, intracardiacally,orally or topically. It will be obvious to those skilled in the art thatthe following dosage forms may comprise as the active ingredient, eithercompounds or a corresponding pharmaceutically acceptable salt of acompound of the present invention. Preferred methods of administrationfor the compounds of the invention include intravenous administrationand oral administration.

For oral administration, the compositions may be in the form of, forexample, a tablet, capsule, suspension or liquid. The composition ispreferably made in the form of a dosage unit containing an effectiveamount of the active ingredient. Examples of such dosage units aretablets and capsules. For therapeutic purposes, the tablets and capsulescan contain, in addition to the active ingredient, conventional carrierssuch as binding agents, for example, acacia gum, gelatin,polyvinylpyrrolidone, sorbitol, or tragacanth; fillers, for example,calcium phosphate, glycine, lactose, maize-starch, sorbitol, or sucrose;lubricants, for example, magnesium stearate, polyethylene glycol,silica, or talc; disintegrants, for example potato starch, flavoring orcoloring agents, or acceptable wetting agents. Oral liquid preparationsgenerally in the form of aqueous or oily solutions, suspensions,emulsions, syrups or elixirs may contain conventional additives such assuspending agents, emulsifying agents, non-aqueous agents,preservatives, coloring agents and flavoring agents. Examples ofadditives for liquid preparations include acacia, almond oil, ethylalcohol, fractionated coconut oil, gelatin, glucose syrup, glycerin,hydrogenated edible fats, lecithin, methyl cellulose, methyl or propylpara-hydroxybenzoate, propylene glycol, sorbitol, or sorbic acid.

The compositions may also be administered parenterally via, for example,injection. Formulations for parenteral administration can be in the formof aqueous or non-aqueous isotonic sterile injection solutions orsuspensions. These solutions or suspensions can be prepared from sterilepowders or granules having one or more of the carriers mentioned for usein the formulations for oral administration. The compounds can bedissolved in polyethylene glycol, propylene glycol, ethanol, corn oil,benzyl alcohol, sodium chloride, and/or various buffers.

Delivery can also be by injection into the brain or body cavity of apatient or by use of a timed release or sustained release matrixdelivery systems, or by onsite delivery using micelles, gels andliposomes. Nebulizing devices, powder inhalers, and aerosolizedsolutions are representative of methods that may be used to administersuch preparations to the respiratory tract. Delivery can be in vitro, invivo, or ex vivo.

The dosage regimen for treating an ischemia, ischemic injury orischemia-reperfusion injury with a compound and/or composition of thisinvention is selected in accordance with a variety of factors, includingthe type, age, weight, sex and medical condition of the subject, theseverity of the ischemia-reperfusion injury, the route and frequency ofadministration, and the particular compound or composition employed. Ingeneral, dosages are determined in accordance with standard practice foroptimizing the correct dosage for treating ischemia-reperfusioninjury-associated disease.

The dosages of a compound of the invention provided to a subject may bevaried depending upon the requirements of the patient, the severity ofthe condition being treated, the route of administration and thecompound being employed. Determination of the proper dosage for aparticular situation is within the skill in the art. For example,suitable dosages for administration to humans can be extrapolated fromdata obtained in experiments performed on animal (e.g., rat) models.Guidance for extrapolating non-human animal model dosage data to humandosages can be found, for example, in FDA Draft Guidance: Estimating theSafe Starting Dose in Clinical Trials for Therapeutics in Adult HealthyVolunteers (2005).

For example, suitable intravenous dosages of a compound of the inventioncan be from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kgto about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about0.01 mg/kg to about 1 mg/kg body weight per treatment. Determining thedosage and route of administration for a particular agent, patient andischemia or ischemia-reperfusion injury is well within the abilities ofone of skill in the art. Preferably, the dosage does not cause orproduces minimal adverse side effects.

An effective amount of a compound of the invention can be administeredalone, or in combination with, one or more other therapeutic agents.Suitable therapeutic agents that are useful for treating ischemicinjuries, which can be administered in combination with a compound ofthe invention, include, but are not limited to, calcium channelblockers, beta blockers, nitroglycerin, aspirin, anti-inflammatoryagents, natriuretic factors, vasodilators, thrombolytic andantithrombolic agents.

Thus, a compound of the invention can be administered as part of acombination therapy (e.g., with one or more other therapeutic agents).The compound of the invention can be administered before, after orconcurrently with one or more other therapeutic agents. In someembodiments, a compound of the invention and other therapeutic agent canbe co-administered simultaneously (e.g., concurrently) as eitherseparate formulations or as a joint formulation. Alternatively, theagents can be administered sequentially, as separate compositions,within an appropriate time frame, as determined by the skilled clinician(e.g., a time sufficient to allow an overlap of the pharmaceuticaleffects of the therapies). A compound of the invention and one or moreother therapeutic agents can be administered in a single dose or inmultiple doses, in an order and on a schedule suitable to achieve adesired therapeutic effect (e.g., a reduction in and/or inhibition ofjoint inflammation; a reduction in and/or inhibition of ischemia, areduction in and/or inhibition of an ischemic injury; a reduction inand/or inhibition of an ischemia-reperfusion injury). Suitable dosagesand regimens of administration can be determined by a clinician and aredependent on the agent(s) chosen, pharmaceutical formulation and routeof administration, various patient factors and other considerations.

One of skill in the art can readily assess the efficacy of a compoundfor treating an ischemic injury or an ischemia-reperfusion injury bymeasuring biochemical or physiological parameters in the subject priorto and after treatment of the subject by using standard assays for theparameter(s) being measured. For example, the efficacy of a compound ofthe invention can be determined by analyzing the levels of surrogatecardiac biomarkers, including certain cardiac enzymes (e.g., creatinekinase (CK-MB), troponin-T, troponin-I), in blood samples obtained froma subject at various time points before and after the ischemic injury orischemia-reperfusion injury, wherein a statistically significantreduction in the levels of the enzymes is indicative of the compoundhaving efficacy in treating the injury. In an exemplary assessment, oneor more blood samples are collected from a subject prior to the injury(e.g., about 6 to about 48 hours prior to the injury) and analyzed forCK-MB and troponin-T levels. Blood samples are then obtained from thesubject at various time points after the injury (e.g., at 6.0, 12.0,18.0, and 24.0 hours after the injury) and CK-MB and troponin-T levelsare analyzed in one or more of these samples.

The efficacy of a compound of the invention can also be determined byelectrocardiogram (ECG) monitoring. For example, standard continuous12-lead ECG monitoring can be performed after fitting the subject with acontinuous 12-lead ECG monitoring device with electronic data storageprior to dosing (e.g., about 5 minutes prior to dosing). The ECGreadings can then be obtained before and after the injury (e.g., untilabout 24 hours after the injury). A change from an abnormal ECG trace toa normal ECG trace (e.g., a reduction of an elevated ST segment) isindicative of the compound having efficacy in treating the injury.

In addition, the efficacy of a compound of the invention can also beassessed by determining the ratio of the myocardial infarct area (MI) tothe ischemic area at risk (AR), according to methods known in the art,wherein a statistically significant reduction of MI/AR ratio isindicative of the compound having efficacy in treating the injury.

EXEMPLIFICATION

Having described the invention generally, the inventors illustrate theinvention with the following examples. These examples are merelyillustrative of certain embodiments of the invention, which is notlimited to exemplified embodiments.

Example 1 Representative Synthesis of Select Compounds of the PresentInvention

Synthesis of RLip-Tau. RLipoic Acid (RLip-OH, 10.0 g) was dissolved inacetone (100 mL, 10 mL/g). The solution was protected from direct lightby covering the reaction flask with foil. N,N-Disuccinimidylcarbonate(15.5 g, 1.25 equivalents) and N,N-diisopropylethylamine (DIEA, 10.5 mL,1.25 equivalents) were added sequentially and the reaction was stirredvented for 2 hours at room temperature to form Lip-OSu in-situ. Taurine(7.0 g, 1.15 equivalents) was added to the solution of Lip-OSu inacetone, followed by the addition of water (50 mL) and DIEA (19.4 mL,2.3 equivalents). The combined solution was stirred overnight.Approximately one third of the crude reaction mixture was transferred toa rotary evaporator and reduced to approximately half volume. Theremaining reaction mixture was injected multiple times directly onto asemi-preparative high-performance liquid chromatography (HPLC) systemand the product isolated on a YMC Pack Pro C18 reverse phase columnusing a gradient of increasing acetonitrile (0.5% acetic acid) in water(0.5% acetic acid). Product-containing fractions were identified byanalytical HPLC, frozen, and lyophilized to provide 2.16 g Lip-Tauat >95% HPLC purity (percent area at 220 nm) as a gummy solid. Theproduct ¹H NMR was consistent with the assigned structure and theproduct had an observed mass of 312 (M−1), calculated 313.

Synthesis of RLip-Tau Lysine salt. The RLip-Tau (2.16 g) isolated bysemi-preparative reverse phase chromatography and lyophilized wasdissolved in 70 mL ethanol. Water (3.0 mL) was added to the ethanolicsolution followed by L-lysine (1.33 g, 1 equivalent). The solution wasshaken overnight, filtered, and rinsed 2 times with 15 mL ethanol.Isolated product was dried under vacuum to yield 3.1 g of the RLip-TauLysine salt with >95% HPLC purity (percent area at 220 nm). The product¹H NMR was consistent with the assigned structure.

The chemical name and structure of exemplary compounds of the inventionare set forth in Table A. Table B contains nuclear magnetic resonance(NMR) data, high performance liquid chromatography (HPLC) data and massspectroscopy data for the compounds in Table A.

TABLE A Chemical Name and Structure of Exemplary Compounds of theInvention Entry Chemical Name Structure A RLip-Taurine-OH

B RLip-Idaa

C RLip-E—OH

D RLip-EG—OH

E (R/S)Lip-DG—OH

F (R/S)Lip-QG—OH

G RLip-EE—OH

H RLip-ES—OH

I (R/S)Lip-HA—OH

J RLip-YA—OH

K RLip-AE—OH

L RLip-Glu-Sar-OH

M RLip-Glu-βAla-OH

N RLip-Glu-Tau-OH

O RLip-DA—OH

P RLip-EE—NH₂

Q RLip-Y—OH

R RLip-βAla-OH

S N-(RLip)-Aminoadipic- OH RLip-Aad

T RLip-D—OH

U RLip-G—OH

V RLip-A—OH

W RLip-γ-aminobutyric acid

X RLip-Phospho-Ser-OH RLip-S(O₃PH₂)—OH

Y RLip-β-Flouro-Ala-OH

Z RLip-DGlu-OH RLip-e-OH

AA SLip-DGlu-OH SLip-e-OH

AB N-(RLip)-4- CarboxyPhe-OH

AC N-(RLip)-3- aminobenzene-sulfonic acid RLip-ABS *Lys

AD N-(RLip)-Sulfanilic- OH RLip-Sulf

AE RLip-Cysteic-OH RLip-Cya *2Lys

AF N-(RLip)-2-aminoethyl hydrogen sulfate RLip-AEHS *Lys

AG N-(RLip)-O- Phosphoryl- ethanolamine RLip-PEA *2Lys

AH N-(RLip)aminoethyl- phosponic acid RLip-AEP *2Lys

AI N-(RLip)-4- aminobenzoic acid RLip-PABA

TABLE B NMR Data, HPLC Data, and Mass Spectroscopy Data of the Compoundsof Table A HPLC Retention Time (min)/Purity Entry ¹H NMR (%) Mass ADithiolane —CH—S— m, 1H, δ 3.12  8.29 Calc: 313 Taurinyl —CH2— m, 2H, δ3.65, t, (100) Found (M − 1): 312 2H, δ 2.98 B Dithiolane —CH—S— m, 1H,δ 3.12 11.4 Calc: 321 Iminodiacetic —CH2— d, 4H, δ 4.20  (90) Found (M −1): 320 C Dithiolane —CH—S— m, 1H, δ 3.05 11.7 Calc: 335 Glutamyl αC—Hm, 1H, δ 4.37  (78) Found (M − 1): 334 D Dithiolane —CH—S— m, 1H, δ 3.11 8.9 Calc: 392 Glutamyl αC—H m, 1H, δ 4.5  (98) Found (M − 1): 391Glycinyl —CH2— d, 2H, δ 3.9 E Dithiolane —CH—S— m, 1H, δ 3.10 10.0 Calc:378 Glutamyl αC—H m, 1H, δ 4.85 (100) Found (M + 1): 379 Glycinyl —CH2—d, 2H, δ 3.9 F NA  9.19 Calc: 391  (99.9) Found (M − 1): 392 GDithiolane —CH—S— m, 1H, δ 3.10 11.0 Calc: 464 Glutamyl, αC—H m, 2H, δ4.40  (97) Found (M + 1): 463 Glutamyl H Dithiolane —CH—S— m, 1H, δ 3.1010.3 Calc: 422 Glutamyl αC—H m, 1H, δ 4.45  (86) Found (M − 1): 421Serinyl αC—H m, 1H, δ 3.90 I NA  9.16/9.29 Calc: 414  (99) Found (M +1): 415 J Dithiolane —CH—S— m, 1H, δ 3.10 13.1 Calc: 440 Alaninyl, αC—Hm, 1H, δ 4.63  (98) Found (M − 1): 439 Tyrosinyl m, 1H, δ 4.40 K NA11.25 Calc: 406  (98) Found (M − 1): 405 L Dithiolane —CH—S— m, 1H, δ3.10 11.4 Calc: 406 Sarcosine —CH2— m, 2H, δ 4.48  (89) Found (M − 1):405 M Dithiolane —CH—S— m, 1H, δ 3.10 10.9 Calc: 406 Glutamyl αC—H m,1H, δ 4.40 (100) Found (M − 1): 405 N Dithiolane —CH—S— m, 1H, δ 3.18 9.05 Cale: 442 Glutamyl αC—H m, 1H, δ 4.2 (100) Found (M − 1): 441Taurinyl —CH2— m, 2H, δ 3.36, t, 2H, δ 2.85 O Dithiolane —CH—S— m, 1H, δ3.05 15.3 Calc: 392 Aspartyl, αC—H m, 1H, δ 4.70  (98) Found (M − 1):391 Alaninyl m, 1H, δ 4.28 P Dithiolane —CH—S— m, 1H, δ 3.05 10.8 Calc:463 Glutamyl, αC—H m, 2H, δ 4.25 (100) Found (M − 1): 462 Glutamyl Q NA12.98 Calc: 369  (95) Found (M + 1): 370 R NA 10.95 Calc: 277 (100)Found (M + 1): 278 S NA 11.23 Calc: 349  (96) Found (M + 1): 350 T NA10.4 Calc: 321  (98) Found (M + 1): 322 U NA 10.7 Calc: 263 (100) Found(M + 1): 264 V NA 11.76 Calc: 277 (100) Found (M + 1): 278 W NA 11.55Calc: 291  (98) Found (M + 1): 292 X NA  8.19 Calc: 373  (96) Found (M +1): 374 Y NA 12.49 Calc: 295 (100) Found (M + 1): 296 Z NA 11.6 Calc:335  (99) Found (M + 1): 336 AA NA 11.6 Calc: 335  (97) Found (M + 1):336 AB NA 12.69 Calc: 397  (99) Found (M + 1): 398 AC NA 12.50 Calc: 361 (95) Found (M − 1): 360 AD NA 12.37 Calc: 361  (92) Found (M − 1): 360AE Dithiolane —CH—S— m, 1H, δ 3.75  7.61 Calc: 359 Lys αC—H m, 1H, δ3.75  (97) Found (M + 1): 360 Cysteic αC—H m, 1H, δ 4.55 AF Dithiolane—CH—S— m, 1H, δ 3.65  9.05 Calc: 329 Lys αC—H t, 1H, δ 3.75 (100) Found(M − 1): 328 AEHS —CH2— t, 2H, δ 4.16 AG Dithiolane —CH—S— m, 1H, δ 3.65 8.32 Calc: 329 Lys αC—H t, 1H, δ 3.75  (89) Found (M − 1): 328 PEA—CH2— q, 2H, δ 3.84 AH Dithiolane —CH—S— m, 1H, δ 3.25  8.63 Calc: 313Lys αC—H t, 1H, δ 3.77  (98) Found (M − 1): 312 Aminoethyl —CH2— m, 2H,δ 2.05 AI NA 15.25 Calc: 325  (99) Found (M + 1): 326

Example 2 Efficacy of Select Lipoyl Compounds of the Invention in a RatModel of MI/AR Injury

Materials and Methods

A rat model of MI/AR injury was used as an in vivo screen to determineif the compounds of Tables 1-5 were cardio-protective (e.g., againstmyocardial ischemia-reperfusion injury). This model is analogous to theischemia-reperfusion injury observed in cardiac patients followingcoronary occlusions and cardiac surgery procedures, such as coronaryartery bypass grafting (CABG) (Matsui, T., Tao, J., del Monte, F., Lee,K.-H. et al., Akt Activation Preserves Cardiac Function and PreventsInjury After Transient Cardiac Ischemia In vivo, Circulation 2001,104:330, the teachings of which are incorporated herein by reference intheir entirety).

General Procedure

The circumflex branch of the left coronary artery (LCA) was ligatedtemporarily to induce regional ischemia in the left ventricular mass,followed by the injection of fluorescent microspheres to delineate theischemic region. 15 minutes prior to ligation (pre-occlusion,pre-ischemic episode), a compound from Tables 1-5 was administered tothe animals. Doses of the compounds listed in Tables 1-5 ranged from1-20 mg/kg. The animals were sacrificed about 24 hours after reperfusionand the hearts were excised, sectioned and stained withtriphenyltetrazolium. The direct impact of the pharmacologicintervention was determined by measuring the myocardial infarct area(MI), the ischemic area at risk (AR) and the left ventricular area (LV).The reduction of MI over the AR (MI/AR ratio) was used as the primarymeasure of drug efficacy relative to vehicle controls. The results arereported in Tables 1-5.

TABLE 1 Di-amino Acids Containing an Acidic Functionality MI/ARReduction Entry Name Structure (%) 1 N-(R)-lipoyl-L- glutamyl-L- alanine(RLip-EA—OH)

31 2 N-lipoyl-L- aspartylglycine (RLip-DG—OH)

28 3 N-(R)-lipoyl-L- tyrosinyl-L- alanine (RLip-YA—OH)

31 4 N-(R)-lipoyl-L- glutamyltaurine (RLip-E-Tau- OH)

33 5 N-(R)-lipoyl-L- alanyl-L- glutamic acid (RLip-AE—OH)

24

TABLE 2 Single Amino Acid Containing Acidic Functionality MI/AR EntryName Structure Reduction (%)  6 N-(R)-lipoyl- L-4-carboxy- phenylalanine(RLip-(4- carboxy)Phe- OH)

26  7 N-(R)-lipoyl- fluoroalanine RLip- fluoroalanine

10  8 N-(R)-lipoyl- L-tyrosine (RLip-Y—OH)

29  9 N-(R)-lipoyl- L-glutamic acid (RLip-E—OH)

33 10 N-(R)-lipoyl- L-cysteic acid RLip-Cya-OH

30 11 N-(R)- lipoyliminodi- acetic acid RLip-Idaa

21

TABLE 3 Alkyl Acids MI/AR Entry Name Structure Reduction (%) 12N-(R)-lipoyl- β-alanine RLip-βAla-OH

39 13 N-(R)-lipoyl- taurine RLip-Tau-OH

45 to 52 14 N-(R)-lipoyl- aminoethyl hydrogen- sulfate

30 15 N-(R)-lipoyl- aminoethyl- phosphonic acid

53 16 N-(R)-lipoyl- aminoethyl dihydrogen- phosphate N-(R)-lipoyl-O-phosphoryl- ethanolamine

47

TABLE 4 Alkyl Bis-Acids MI/AR Entry Name Structure Reduction (%)  9N-(R)-lipoyl-L- glutamic acid (RLip-E—OH)

33 10 N-(R)-lipoyl-L- cysteic acid RLip-Cya-OH

30 11 N-(R)- lipoyliminodi- acetic acid RLip-Idaa

21

TABLE 5 Aromatic Acids MI/AR Entry Name Structure Reduction (%) 17N-(R)-lipoyl- para- aminobenzoic acid (RLip-PABA)

22  6 N-(R)-lipoyl- L-4-carboxy- phenylalanine (R)Lip-(4- carboxy)F—OH

26 18 N-(R)-lipoyl- meta- aminobenzene sulfonic acid

33 19 N-(R)-lipoyl- para- aminobenzene sulfonic acid N-(R)-lipoyl-sulfanilic acid

44  8 N-(R)-lipoyl-L- tyrosine (RLip-Y—OH)

29Detailed Procedure

Male Sprague-Dawley rats between 300 and 350 g were used for theseexperiments. Anesthesia was induced with 3-4% isoflurane in an inductionchamber. After induction, anesthesia was maintained at a surgical planewith 1.5-2.0% isoflurane, administered by a Rodent Ventilator through a16-gauge angiocatheter introduced orally into the trachea. Theventilator was set at 2.5 cc at a rate of 60-65 breaths per minute tomaintain ventilation during surgery. The core temperature of the animalwas monitored and maintained at 37° C. using a rectal probe and aheating lamp attached to a temperature controller.

A left anterior thoracotomy was performed and the heart was exposedusing a vertical pericardotomy. The circumflex branch of the leftcoronary artery (LCx) was ligated approximately 4 mm from the aortausing a cardiovascular 7.0 monofilament suture on an 11 mm needle toinduce ischemia in the left ventricle.

Fluorescent microspheres (300 μL) were injected into the leftventricular cavity 10-20 minutes after the ligation to delineate theischemic area. The suture was removed 30 minutes after ligation toreperfuse the ischemic area and the ischemic area was checked forreperfusion.

The chest was then closed in layers using absorbable suture (Dexon 5-0)for the muscle layers and monofilament Nylon 5-0 suture was used toclose the cutaneous layer. The animals were allowed to recover, thenwere returned to the colony.

Twenty-four hours after reperfusion, anesthesia was induced usingketamine hydrochloride and the chest was opened. The animals weresacrificed with 15% potassium chloride aqueous solution (w/v) injectedinto the LV cavity to arrest the heart in diastole. The heart wasexcised distal to the aortic valve and washed with saline to remove theblood. Sagittal slices of the heart were obtained between the base ofthe ventricle and the apex. Five slices of heart tissue were obtained,each 2 mm thick. The slices were immersed in a 1%2,3,5-triphenyl-2H-tetrazolium chloride (TTC) in saline solution andthen stored in the dark for 30 minutes to stain.

Images of the slices were obtained under bright field (to observe theTTC staining) and under fluorescence (to observe the microspheres). Thearea at risk was determined by the absence of microspheres and theinfarct area was determined by the absence of TTC staining.

Results

The compounds of Tables 1-5, administered as an intravenous injection,effectively reduced the myocardial infarct (MI) size relative to thearea at risk (AR). A significant reduction in the area of cardiac damagewas observed in myocardial tissue sections following treatment with acompound of Tables 1-5.

Example 3 Efficacy of Select Lipoyl Compounds of the Invention in a PACRat Model of Ischemia-Induced Renal Injury

Materials and Methods

A partial aortic clamping (PAC) rat model of ischemia-induced renalinjury was used as an in vivo screen to determine if RLip-EA-OH (Entry1), RLip-Cya-OH (Entry 10), RLip-Tau (Entry 13), andRLip-aminoethylphosphonic acid (Entry 15) were renal-protective (e.g.,against renal ischemia-reperfusion injury). This model simulatesischemia-reperfusion injury observed in renal patients followingischemia-induced renal failure (Molitoris, B. A., Dagher, P. C.,Sandoval, R. M., Campos, S. B., Ashush, H., Fridman, E., Brafman, A.Faerman, A., Atkinson, S. J., Thompson, J. D., Kalinski, H., Skaliter,R., Erlich, S., Feinstein, E. “siRNA Targeted to p53 Attenuates Ischemicand Cisplatin-Induced Acute Kidney Injury.” J Am Soc Nephrol, 2009, vol.20, 1754-1764, the teachings of which are incorporated herein byreference to their entirety). Serum creatinine concentrations (SCr)typically rise due to renal ischemia and effective treatment should showa reduction in serum creatinine concentrations. Reduction in serumcreatinine concentrations after renal ischemia induction indicates thatthe administered compound is has a protective effect and is effective inreducing ischemia-induced kidney injury.

General Procedure

The abdominal aorta just below the renal arteries was isolated andtemporarily ligated to induce regional ischemia using an aortic clamp.An initial blood sample was drawn at study initiation for baselinecreatinine measurement and at 24 hours post-surgery for functionalevaluation of the severity of kidney injury. The animals were sacrificedabout 24 hours after reperfusion.

Detailed Procedure

Male Sprague-Dawley rats between 200 to 250 g were used for theseexperiments. Anesthesia was induced with 5% halothane and maintainedwith 1-1.5% halothane in oxygen-enriched air via a face mask. The ratswere maintained on a warming blanket throughout the procedure tomaintain body temperature at 37° C. After shaving the abdomen of therat, a midline incision was made through the skin and musculature toexpose the abdominal cavity to quantify the aortic blood flow (ABF).

The abdominal aorta just below the renal arteries was isolated throughblunt dissection from the inferior vena cava, and an ultrasonic probe(2.0-mm diameter, Transit Time Perivascular Flowmeter TS420) was placedand secured. The upper abdominal aorta was then isolated through bluntdissection and freed from the surrounding structures to expose the aortabetween the celiac artery and superior mesenteric artery.

Aortic clamps comprised of two 4-mm in length polyethylene tubes(PE-100, 0.86-mm diameter) were then placed around the aorta to inducerenal ischemia. One clamp was placed around the aorta and the other wasplaced to exert variable tension via a 10-in. 3.0 silk suture. The silkthread was then tied and the tension on the two ends of the threadincreased until there was a 90% reduction of initial (ABF) rate measuredon the ultrasonic probe reader. A 10% baseline blood flow was maintainedfor a period of 30 minutes.

RLip-EA-OH (Entry 1) at 10 mg/kg; RLip-Cya-OH (Entry 10) at 3 mg/kg;RLip-Tau (Entry 13) at 3 mg/kg; and RLip-aminoethylphosphonic acid at 10mg/kg, 3 mg/kg, and 1 mg/kg were administered intravenously via thefemoral vein as a bolus dose 15 minutes prior to ischemia. A 0.15-mLvenous blood sample was drawn at study initiation for baselinecreatinine measurement and at 24 hours postsurgery for functionalevaluation of the severity of kidney injury. Serum creatinineconcentrations were then measured on a Creatinine Analyzer 2. Theefficacy of RLip-EA-OH and RLip-Tau were measured against the results ofvehicle-treated animals in the blinded studies. A 2-tail t-test was usedto determine differences between treatments.

Results

RLip-EA-OH (Entry 1), RLip-Cya-OH (Entry 10), RLip-Tau (Entry 13), andRLip-aminoethylphosphonic acid (Entry 15), administered intravenously,effectively reduced serum creatinine concentrations relative to thecontrol animals, as shown in Table 6. This reduction in SCr followingtreatment indicated that RLip-EA-OH, RLip-Cya-OH, RLip-Tau, andRLip-aminoethylphosphonic acid had protective effects and minimizedrenal ischemic injury. The data for reduction in cardiac injury reportedin the last column in Table 6 were obtained using the proceduredescribed in Example 2.

TABLE 6 Effects of compounds on renal ischemia reperfusion injury in PACrat model and heart ischemia reperfusion injury in LCA rat model.Reduction of Reduction in Dose Group SCr vs. control Cardiac injuryVehicle (Meta) — — Lip-EA 39.5% 33% 10 mg/kg Lip-Tau 39.5% 50%  3 mg/kgLip-Cya-OH 39.5% 30%  3 mg/kg Lip-aminoethyl 76.0% 32% phosphonic acid10 mg/kg Lip-aminoethyl 28.6% 50% phosphonic acid  3 mg/kgLip-aminoethyl 28.6% 30% phosphonic acid  1 mg/kg

The relevant teachings of all patents, published applications andreferences cited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A composition comprising: (i) a compoundrepresented by Structural Formula (I):

or a pharmaceutically acceptable salt or prodrug thereof, wherein: theenantiomeric excess or diastereomeric excess of the compound orpharmaceutically acceptable salt or prodrug thereof is at least or about90%; R is (C₁-C₁₈)alkyl, (C₆-C₁₈)aryl or (C₆-C₁₈)aryl(C₁-C₁₈)alkyl andis substituted with at least one acidic substituent selected from thegroup consisting of —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H, —OPO₃H₂, —B(OH)₂ and—NHOH, wherein the aryl of the (C₆-C₁₈)aryl or (C₆-C₁₈)aryl(C₁-C₁₈)alkylis optionally further substituted with one or more substituents selectedfrom the group consisting of hydroxy, halo, (C₁-C₃)alkyl,halo(C₁-C₃)alkyl, cyano, nitro, (C₁-C₃)alkoxy and thio(C₁-C₃)alkyl; R′is hydrogen or (C₁-C₁₈)alkyl, wherein (C₁-C₁₈)alkyl is optionallysubstituted with one or more acidic substituents selected from the groupconsisting of —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H, —OPO₃H₂, —B(OH)₂ and —NHOH;and X is absent, provided that the compound of Structural Formula (I) isnot N—(R)-lipoyl-aminoethylphosphonic acid or(R)-5-(5-(1,2-dithiolan-3-yl)pentanamido)-2-hydroxybenzoic acid; and(ii) a pharmaceutically acceptable carrier or diluent.
 2. Thecomposition of claim 1, wherein R is (C₁-C₃)alkyl.
 3. The composition ofclaim 1, wherein R is (C₆)aryl.
 4. The composition of claim 1, wherein Ris (C₆)aryl(C₁-C₃)alkyl.
 5. The composition of claim 2, wherein R is(C₁-C₃)alkyl substituted with one or two acidic substituents eachindependently selected from —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H and —OPO₃H₂. 6.The composition of claim 1, wherein R′ is hydrogen.
 7. The compositionof claim 6, wherein R is (C₁-C₃)alkyl substituted with one or two acidicsubstituents each independently selected from —CO₂H, —SO₃H, —PO₃H₂,—OSO₃H and —OPO₃H₂.
 8. The composition of claim 6, wherein R is(C₆)aryl(C₁-C₃)alkyl substituted with one or two acidic substituentseach independently selected from —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H and—OPO₃H₂, and wherein aryl is optionally substituted with halo andhydroxy.
 9. The composition of claim 8, wherein R is (C₂)alkylsubstituted with one or two acidic substituents each independentlyselected from —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H and —OPO₃H₂.
 10. Thecomposition of claim 8, wherein R is (C₆)aryl substituted with oneacidic substituent selected from —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H and—OPO₃H₂.
 11. The composition of claim 1, wherein R′ is (C₁-C₃)alkyl. 12.The composition of claim 1, wherein R and R′ are each (C₁-C₃)alkylsubstituted with one acidic substituent selected from —CO₂H, —SO₃H,—PO₃H₂, —OSO₃H and —OPO₃H₂.
 13. The composition of claim 1, wherein thepharmaceutically acceptable salt comprises a monovalent cation or adivalent cation.
 14. The composition of claim 1, wherein thepharmaceutically acceptable salt comprises an amine.
 15. The compositionof claim 1, wherein the pharmaceutically acceptable salt compriseslysine.
 16. The composition of claim 1, wherein the compound isrepresented by one of the following structural formulas:

or a pharmaceutically acceptable salt of any of the foregoing.
 17. Amethod for treating an ischemic injury or an ischemia-reperfusion injuryin a subject in need thereof, comprising administering to the subject aneffective amount of a composition comprising a compound represented byStructural Formula (I):

or a pharmaceutically acceptable salt or prodrug thereof, wherein: theenantiomeric excess or diastereomeric excess of the compound orpharmaceutically acceptable salt or prodrug thereof is at least or about90%; R is (C₁-C₁₈)alkyl, (C₆-C₁₈)aryl or (C₆-C₁₈)aryl(C₁-C₁₈)alkyl andis substituted with at least one acidic substituent selected from thegroup consisting of —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H₂, —OPO₃H₂, —B(OH)₂ and—NHOH, wherein the aryl of the (C₆-C₁₈)aryl or (C₆-C₁₈)aryl(C₁-C₁₈)alkylis optionally further substituted with one or more substituents selectedfrom the group consisting of hydroxy, halo, (C₁-C₃)alkyl,halo(C₁-C₃)alkyl, cyano, nitro, (C₁-C₃)alkoxy and thio(C₁-C₃)alkyl; R′is hydrogen or (C₁-C₁₈)alkyl, wherein (C₁-C₁₈)alkyl is optionallysubstituted with one or more acidic substituents selected from the groupconsisting of —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H, —OPO₃H₂, —B(OH)₂ and —NHOH;and X is absent, provided that the compound of Structural Formula (I) isnot N—(R)-lipoyl-aminoethylphosphonic acid or(R)-5-(5-(1,2-dithiolan-3-yl)pentanamido)-2-hydroxybenzoic acid.
 18. Themethod of claim 17, wherein R is (C₁-C₃)alkyl.
 19. The method of claim17, wherein R is (C₆)aryl.
 20. The method of claim 17, wherein R is(C₆)aryl(C₁-C₃)alkyl.
 21. The method of claim 17, wherein R is(C₁-C₃)alkyl substituted with one or two acidic substituents eachindependently selected from —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H and —OPO₃H₂.22. The method of claim 17, wherein R′ is hydrogen.
 23. The method ofclaim 22, wherein R is (C₁-C₃)alkyl substituted with one or two acidicsubstituents each independently selected from —CO₂H, —SO₃H, —PO₃H₂,—OSO₃H₂ and —OPO₃H₂.
 24. The method of claim 22, wherein R is(C₆)aryl(C₁-C₃)alkyl substituted with one or two acidic substituentseach independently selected from —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H and—OPO₃H₂, and wherein aryl is optionally substituted with halo orhydroxy.
 25. The method of claim 22, wherein R is (C₂)alkyl substitutedwith one or two acidic substituents each independently selected from—CO₂H, —SO₃H, —PO₃H₂, —OSO₃H and —OPO₃H₂.
 26. The method of claim 22,wherein R is (C₆)aryl substituted with one acidic substituent selectedfrom —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H and —OPO₃H₂.
 27. The method of claim17, wherein R′ is (C₁₋₃)alkyl.
 28. The method of claim 17, wherein R andR′ are each (C₁-C₃)alkyl substituted with one acidic substituentselected from —CO₂H, —SO₃H, —PO₃H₂, —OSO₃H and —OPO₃H₂.
 29. The methodof claim 17, wherein the ischemia-reperfusion injury is selected fromthe group consisting of a cerebrovascular ischemia-reperfusion injury, arenal ischemia-reperfusion injury, a hepatic ischemia-reperfusioninjury, an ischemia-reperfusion cardiomyopathy, a cutaneousischemia-reperfusion injury, a bowel ischemia-reperfusion injury, anintestinal ischemia-reperfusion injury, a gastric ischemia-reperfusioninjury, a pulmonary ischemia-reperfusion injury, a pancreaticischemia-reperfusion injury, a skeletal muscle ischemia-reperfusioninjury, an abdominal muscle ischemia-reperfusion injury, a limbischemia-reperfusion injury, ischemia-reperfusion colitis, a mesentericischemia-reperfusion injury and a silent ischemia-reperfusion injury.30. The method of claim 17, wherein the ischemic injury is a myocardialischemic injury.
 31. The method of claim 17, wherein the ischemic injuryis consequent to an ischemia selected from the group consisting of acardiovascular ischemia, a cerebrovascular ischemia, a renal ischemia, ahepatic ischemia, a ischemia-reperfusion cardiomyopathy, a cutaneousischemia, a bowel ischemia, an intestinal ischemia, a gastric ischemia,a pulmonary ischemia, a pancreatic ischemia, a skeletal muscle ischemia,an abdominal muscle ischemia, a limb ischemia, an ischemia-reperfusioncolitis, a mesenteric ischemia and a silent ischemia.
 32. The method ofclaim 17, wherein the ischemia-reperfusion injury is a myocardialischemia-reperfusion injury.
 33. The method of claim 32, wherein themyocardial ischemia-reperfusion injury is consequent to a myocardialinfarction.
 34. The method of claim 33, wherein the myocardialinfarction is an acute myocardial infarction.
 35. The method of claim17, wherein the ischemia-reperfusion injury is a cerebralischemia-reperfusion injury.
 36. The method of claim 35, wherein thecerebral ischemia-reperfusion injury is consequent to a stroke.
 37. Themethod of claim 17, wherein the ischemic injury or ischemia-reperfusioninjury includes peri-operative cardiac damage.
 38. The method of claim17, wherein the ischemia-reperfusion injury is a renalischemia-reperfusion injury.
 39. The method of claim 17, wherein theischemic injury or ischemia-reperfusion injury is consequent to atherapeutic intervention.
 40. The method of claim 17, wherein thetherapeutic intervention is selected from the group consisting of acoronary artery bypass graft surgery, a coronary angioplasty surgery, atransplant surgery and a cardiopulmonary bypass surgery.
 41. The methodof claim 17, wherein the compound or pharmaceutically acceptable saltthereof is administered to the subject orally.
 42. The method of claim17, wherein the compound or pharmaceutically acceptable salt thereof isadministered to the subject intravenously.
 43. The method of claim 17,wherein the compound is represented by any one of the followingstructural formulas:

or a pharmaceutically acceptable salt of any of the foregoing.
 44. Thecomposition of claim 1, wherein the enantiomeric excess ordiastereomeric excess of the compound or pharmaceutically acceptablesalt or prodrug thereof is at least or about 99%.
 45. The method ofclaim 17, wherein the enantiomeric excess or diastereomeric excess ofthe compound or pharmaceutically acceptable salt or prodrug thereof isat least or about 99%.